Lachnea poiraultii (), rediscovered after more than one hundred years

Nicolas VAN VOOREN Summary: Recent collections of a cup- identified asLachnea poiraultii Boud. from the Iberian and Is- Uwe LINDEMANN trian (Croatia) peninsulas provided the opportunity to investigate its modern taxonomic position. An exa- Marcel VEGA mination of the type specimen of this taxon confirmed the determination of Croatian, Portuguese and Miguel Ángel RIBES Spanish collections. Based on the results of multi-gene phylogenetic analyses which placed L. poiraultii within the with no support of affiliation with another genus, and the study of microscopic features Tomás ILLESCAS in the living state, the new genus Paratricharina is proposed to accommodate L. poiraultii. The morphological Neven MATOČEC similarities and differences from the most closely related are discussed. A full description and illus- Ivana KUŠAN trations of P. poiraultii are also provided. Keywords: , Pyronemataceae, Tricharina, Paratricharina, , phylogeny. Ascomycete.org, 7 (3) : 105-116. Juin 2015 Résumé : de récentes récoltes d’un discomycète identifié comme Lachnea poiraultii Boud. en provenance Mise en ligne le 13/06/2015 des péninsules ibériques et istriennes (Croatie) ont fourni l’opportunité d’évaluer son placement taxinomique moderne. Un examen de spécimens type de ce taxon a confirmé la détermination des récoltes croates, por- tugaises et espagnoles. Sur la base des résultats d’une analyse phylogénétique multigènes qui ont placé L. poiraultii au sein des Pyronemataceae sans support d’un rattachement à un autre genre, et l’étude des ca- ractères microscopiques à l’état vivant, le nouveau genre Paratricharina est proposé pour accommoder L. poi- raultii. Les similitudes morphologiques et les différences avec les espèces les plus proches sont discutées. Une description complète et des illustrations de P. poiraultii sont également fournies. Mots-clés : Ascomycota, Pyronemataceae, Tricharina, Paratricharina, taxinomie, phylogénie.

Introduction ween spore length and width, the value in italics represents the mean value of this ratio. The terminology and abbreviations of cytological elements (cell Lachnea poiraultii was published by BOUDIER (1901) to accommo- date an operculate discomycete with an orange hymenium, hairy inclusions and exudates) related to the living cells are used after outer surface and microscopic characters similar to those of the BARAL (1992). Spore shape is given after KUŠAN et al. (2014). genus Tricharina Eckblad. Boudier included it in the genus Lachnea Macrophotographs were made in situ using digital cameras, while in his own sense (BOUDIER, 1885, 1907), i.e. a group of species in the microphotographs have been taken using digital cameras mounted Pezizales with stiff brown hairs, longer at the margin, and having directly on microscopes. Line drawings were made freehand to smooth or more rarely verrucose ascospores containing guttules. scale. No other collection of this species has been reported since its first description, for more than one hundred years. In the revision of Bou- DNA extraction, amplification and sequencing dier’s Icones by KORF (1985), this taxon was considered as a putative Geopora Harkn. or Fuckel species. Total DNA was extracted from dry specimens blending a portion Recent collections made in Croatia, Portugal and Spain, the exa- of them with the aid of a micropestle in 600 μl CTAB buffer (CTAB mination of type specimens, and phylogenetic analyses of multiple 2%, NaCl 1.4 M, EDTA pH 8.0 20 mM, Tris-HCl pH 8.0 100 mM). The DNA loci facilitated a re-circumscription of this species and a pro- resulting mixture was incubated for 15 min at 65 ºC. A similar vo- posal for its taxonomic placement. lume of chloroform: isoamyl alcohol (24:1) was added and carefully mixed with the samples and emulsified. It was then centrifuged for Materials and Methods 10 min at 13,000 g, and the DNA in the supernatant was precipitated with a 1/1 volume of isopropanol. After a second centrifugation for Morphology, cytology and cytochemistry 15 min at the same speed, the pellet was washed in cold 70% etha- The observations were made on fresh and dried material using nol, centrifuged again for 2 min and dried. It was finally resuspended methods of “vital taxonomy” (BARAL, 1992) supplemented with cy- in 200 μl of ddH2O. PCR amplification was performed with the pri- tological and cytochemical tests using lethal media applied directly mers ITS1F and ITS4 (WHITE et al., 1990; GARDES & BRUNS, 1993) for ITS, to the living cells; some small pieces of dried specimens were rehy- LR0R and LR5 (VILGALYS & HESTER, 1990) were used to amplify the 28S drated during about twelve hours in water. The following mounts nLSU region, EF1-983F and EF1-1567R (REHNER & BUCKLEY, 2005) for were used to observe microscopic characters: water, 0.5 and 5% the translation elongation factor 1α tef1 gene, and reverse of bRPB2- KOH, iodine reagents (Lugol’s solution, Melzer’s reagent), Methyl 6R2 (MATHENY et al., 2007) and a modified version of fRPB2-7.1R (MA- (Cotton) Blue both in lactophenol and lactic acid, and 1% aqueous THENY, 2005), fRPB2-7.1R2 (5’ – CCCATNGCYTGYTTVCCCATDGC – 3’) solution of CRB (Cresyl Blue Brilliant), Congo Red (CR, after PFISTER et for the RNA polymerase II second largest subunit, rpb2. PCR reac- al., 2009). Measurements were made on ≥ 20 ascospores mounted tions were performed under a program consisting of a hot start at in water from each collection, except on Croatian material where 50 95 ºC for 5 min, followed by 35 cycles at 94 ºC, 54 ºC and 72 ºC (45, living ascospores mounted in tap water were measured and 50 dead ascospores mounted in Methyl blue in lactic acid (in two Croatian 30 and 45 s respectively) and a final 72 ºC step 10 min. PCR products collections, both from spore deposits) to test the shrinkage of dead were checked on 1% agarose gels (visualized with GelRed dye), and spores. Spores are measured under the 100× oil immersion lens of positive reactions were sequenced with one or both of the sequen- transmission light microscopes, excluding the ornamentation. X re- cing primers. Chromatograms were checked in MEGA 5.0 (TAMURA et presents the mean value of spore dimensions, and Q the ratio bet- al., 2011) for putative reading errors, and these were corrected. 105 Phylogenetic analyses (15.0) 15.8–17.5 (18.2) × (8.1) 8.8–9.4 (10.3) μm [X=16.6 × 9.2 μm, BLAST was used to select the most closely related sequences from Q=1.6–1.8–2.0] in dead state from Methyl Blue/lactic acid mount, the International Nucleotide Sequence Database Collaboration hyaline, equatorially uninucleate, appearing non-guttulate in dead (INSDC) public databases. The selected sequences came mainly state (lipid bodies masked due to increased refractivity of collapsed from HANSEN et al. (2005a), HANSEN & PFISTER (2006), PERRY et al. (2007), sporoplasm) but with minute bipolar dispersed lipid aggregations HANSEN et al. (2013), and STIELOW et al. (2013), including those of Gla- that rapidly coalesce to form biguttulate lipid pattern in living state, ziella aurantiaca (Berk. & M.A. Curtis) Sacc., chosen as outgroup. In- more or less thick-walled, smooth observed in water and Cotton trons were removed from tef1 and rpb2 data sets. Sequences were Blue, but with a rough to finely verrucose perispore when mounted first aligned in MEGA 5.0 (TAMURA et al., 2011) software with its Clustal in Lugol’s solution, entirely encapsulated by delicate but persistent W application and then corrected manually. The final alignment in- sticky sheath best visible in CR mount. cluded 331/848 (28S nLSU), 355/867 (tef1) and 311/597 (rpb2) va- riable sites. The concatenated alignment was loaded in PAUP* Studied collections: CROATIA. Brtonigla-Nova Vas (Istra County), 4.0b10 (SWOFFORD, 2001) and each locus was subjected to MrModel- N 45.372144°, E 13.632355°, 112 m asl, 30 March 2013, leg. M.J. Šimić, I. Kušan & N. Matočec, CNF-2/9332 and 27 February 2014, leg. M.J. test 2.3 (NYLANDER, 2004). The Model GTR+Γ+I was selected for all par- Šimić, CNF-2/9560, both collected on sandy moist soil densely beset titions, and implemented in MrBayes 3.1 (RONQUIST & HUELSENBECK, 2003), where a Bayesian analysis was performed (LSU-tef1-rpb2 data by mosses along small stream in degraded thermophilic deciduous partitioned, two simultaneous runs, six chains, with temperature set forest of Ulmus canescens, Prunus spinosa and Rubus sp. PORTUGAL. to 0.2, and sampling every 100th generation) until convergence pa- Serra de Montejunto, N 39.18061°, E -9.02545°, 188 m asl, 24 January rameters were met after about 260,000 generations at which point 2014, leg. M. Vega, on bare ground, on a narrow footpath; pers. herb. the standard deviation had fallen below 0.01. The initial 25% of sam- M.V. 20140124-04, U.L. 178-14. SPAIN. Jaén, Aldeaquemada, Río pled trees were discarded as burn-in. In addition to Bayesian analy- Guarrizas, N 38.40786°, E -3.383139°, 700 m asl, 16 March 2014, leg. sis, a full search for the best-scoring maximum likelihood tree was T. Illescas, M. Á. Ribes et al., on naked soil, with some little mosses, close to a river; pers. herb. N.V. 2014.03.24, U.L. 179-14. Málaga, Jar- performed in RAxML (STAMATAKIS, 2006) using the standard search al- dín Botánico-Histórico La Concepción, N 36.76587°, E -4.42523°, gorithm (LSU-tef1-rpb2 data partitioned), the same model of nu- 90 m asl, 24 January 2015, leg. and det. M. Vega, conf. U. Lindemann, cleotide substitution, and node support evaluated by 2,000 on bare ground below Olea sp. between mosses (Barbula sp., Dicra- bootstrap replications. Significance threshold was set at or above nella sp., Bryum sp., Tortula sp.); pers. herb. M.V. 20150124-01. Same 0.95 for posterior probability (PP) and 65% bootstrap proportions locality, N 36.76608°, E -4.42456°, 86 m asl, 24 January 2015, leg. and (BP). Sequences generated during this study were deposited in Gen- det. M. Vega, conf. U. Lindemann, next to a small track on soil, bet- bank under the accession numbers listed in Table 1. ween mosses (Barbula sp., Dicranella sp.); pers. herb. M.V.20150124- 02. Taxonomy Other collection: SPAIN. Barcelona, Les Fonts de Terrassa, Can Fo- Description of recent collections nellet, N 41.51940°, E 2.03369°, 172 m asl, 29 January 2015, leg. and Apothecia 5–25 (40) mm in diameter, sessile, slightly cupulate det. J. Bometón, conf. M.Á. Ribes, on a slope, on soil between with inrolled margin, expanded at the end in Iberian collections, first mosses, with Cupressus sempervirens, Ulmus minor and Prunus sp. with a dull orange hymenium, then becoming brick-coloured, but occasionally slightly yellowish or cream-coloured (the pigment Type material revision seems to be soluble in water); outer surface covered by dense brown-red hairs, often agglutinated to form a brown pustulate pat- There are three collections named Lachnea poiraultii in Boudier’s tern, sometimes appressed, but mostly straight and projecting out- herbarium housed in Muséum national d’histoire naturelle (PC); it wards. Subhymenium thin, 50–70 μm thick, of short-celled textura seems that they were all collected in the same locality in southern intricata to intricata-epidermoidea, with pale orange hyphae. Me- France, although one of them is provided without location or date: dullary excipulum 200–250 μm thick, of textura intricata, with hya- • PC0738324: Lachnea Poiraulti Boud. Icon. mycol. n° 568, Antibes line and thin-walled hyphae, predominantly horizontal in upper ad terram Februario 1900 misit D. Poirault; portion, gradually changing its orientation towards the ectal exci- • PC0738328: Lachnea Poiraulti n° 17, no date, no location, accom- pulum. Ectal excipulum 230–290 μm thick, of textura prismatica, panied by a drawing of spores; with predominantly vertically oriented cells in upper portion, 7– • PC0738329: Lachnea Poiraulti Boud., Antibes, Villa Thuret, Février 25 μm wide, hyaline, becoming a textura globulosa-subangularis in 1900 misit D. Poirault. the outermost part, with brownish cells, measuring (16) 20–41 (52) μm wide. Marginal hairs organized in more or less triangular Following the original description by BOUDIER (1901: 199-200), this bundles; hairs of two types: short, 30–70 × 8–10 μm, obtuse; or long, species has been found “à Antibes, dans le parc de la Villa Thuret [...] 95–270 × 7–12 μm, obtuse or almost pointed at the top; all of them par notre collègue et ami M. G. Poirault.” The diagnosis indicates “Fe- are pale brown, more or less sinuous, septate, with wall 1.8–2.2 μm bruario 1900” as the date. The collections PC0738324 and thick, and with a simple base arising from the globose cells of the PC0738329 agree with these data, but it is not possible to ascertain outer layer of the ectal excipulum. Excipular hairs similar to those whether they are duplicates or two different collections from the on the margin, but longer, reaching 450 μm in length, more or less same locality. We propose to designate the collection PC0738324 flexuous.Asci cylindrical, 300–350 × (11) 14–17.5 (19) μm, 335–382 as the lectotype. × 17.3–19.8 μm when fully turgescent (living state), shortly bifurcate Here is the microscopic description of the revised collection: at the base, arising from perforated croziers, inamyloid, 8-spored. Medullary excipulum of textura intricata. Ectal excipulum of tex- Paraphyses cylindrical, 4–7 μm at the top, without reaction in tura angularis, with cells up to 40 μm wide, with some compact Lugol’s solution or Melzer’s reagent; in the living state apical cell brown cells in the outermost part. Excipular hairs rather dense, 90– contains large hyaline refractive globular to oblong KOH-soluble cy- 420 × 7–12.5 μm, obtuse or slightly pointed, brown, septate, with toplasmic bodies, large non-refractive vacuoles that may contain wall 1.5–3 μm wide, partially enlarged at the base which is simple. single or few minute brick-orange carotenoid granule(s) mixed with Anchoring hyphae present at the base of the apothecia, up to hyaline, minute weakly refractive vacuolar bodies. Ascospores nar- 750 μm in length. Marginal hairs present but broken and hard to rowly ellipsoid, (13.5) 15–19.8 (20.1) × 9–11 (11.3) μm [X=17.8 × evaluate. Asci cylindrical, about 300 μm in length and 10–11 μm 10.2 μm, Q=1.4–1.8–2.0] when freshly ejected in living state and wide, with crozier, inamyloid, 8-spored. Paraphyses cylindrical, 106 Plate 1 — Paratricharina poiraultii A-B. Collection from Jaén (Spain). C. Collection from Serra de Montejunto (Portugal). D. Lectotype of Lachnea poiraultii. E-F. Collections from Málaga (Spain). G. Habitat of the collections from Jardín Botánico-Histórico La Concepción at Málaga. Photos: A-B: M.A. Ribes; C, E-G: M. Vega; D: N. Van Vooren (with the agreement of MNHN). 107 Plate 2 — Paratricharina poiraultii. Microscopic features A. Vertical section of an apothecium. B-C. Excipulum showing a textura prismatica. D. Cells close the margin. E-G. External and marginal hairs. H. Asci and paraphyses. I. Content of paraphyses. J. Living ascospores in water. K. Ascospores in Lugol’s solution, showing an orna- mented perispore. L. Ascospores in Cotton Blue. M. Ascospores showing a refractive layer. N. Perispores. O. Ascospores in Lugol’s solution. A, E, G, K, M and N from U.L. 178-14; B-D, F, H-J, L and O from U.L. 179-14. Scale bars = 10 μm. Photos: U. Lindemann. 108 Plate 3 — Paratricharina poiraultii. Apothecium and texture A: Apothecium in situ (bar = 1cm). B: Apothecial vestiture (bar = 200 μm). C: Marginal texture in original spatial position (bar = 50 μm). D: Excipular texture at middle flank (bar = 50 μm). All from CNF 2/9332. Photos and drawings: N. Matočec. 109 about 3 μm wide at the apex, hyaline. Ascospores ellipsoid, (16) 17– As stated in the discussion and in the phylogenetic analyses, both 20 (21) × (9.2) 9.5–10.5 (11) μm [X = 18.8 × 10.1 μm, Q=1.7–1.9–2.0], microscopic and macroscopic characters of Lachnea poiraultii coun- hyaline, smooth, rather thick-walled, with some polar inclusions that ter a classification of this fungus in the genusTricharina in the sense can merge and produce two polar guttules. of ECKBLAD (1968) and YANG & KORF (1985). Furthermore, Tricharina appears to be paraphyletic and requires additional investigation to Nomenclatural considerations clarify the systematic position of its type-species, T. gilva (Boud.) Eck- blad, within the Geopora clade.

The genus Lachnea (Fr.) Boud. is illegitimate because Lachnea L. Based on our evidences (see Discussion), we propose the new (1753) has priority. As typified by BOUDIER (1885) and confirmed by genus Paratricharina: ECKBLAD (1968: 158), the genus falls in synonymy with Humaria Paratricharina Van Vooren, U. Lindemann, M. Vega, Ribes, Illescas Fuckel, with Peziza hemisphaerica Hoffm. as type-species — which & Matočec, gen. nov. — MB 812169 binomial is thus synonymized as Humaria hemisphaerica (Hoffm.) Description: Apothecia about 10–20 mm in diameter or less, ses- Fuckel. The phylogenetic and morphological evidence presented sile, slightly cupulate with inrolled margin, expanded when old; here do not support L. poiraultii as a Humaria. outer surface covered by dense brown-red hairs, sometimes appres-

Plate 4 — Paratricharina poiraultii. Hymenial elements A: Paraphysis apical cell (in statu vivo); 1- carotenoid granular pigment; 2- non-refractive vacuole; 3- SCB; 4- minute VB; 5- exudate. B: Ascus apex in tap water (in statu vivo). C: Ascus apex in CRB (in statu vivo). D: Ascus apex in CR aqueous solution (in statu vivo, spores not shown). E: Ascal bases with a part of the ascogenous system in tap water (in statu vivo). F: Ascal bases with a part of the ascogenous system in Cotton Blue/lactic acid (in statu emortuo). G: Three freshly ejected ascospores in tap water (in statu vivo); note rapid coalescence of lipid bodies from bipolar dispersed configuration into biguttulate pattern and onstantc encapsulation by a sheath. H: Ascospore in Cotton Blue/lactic acid (in statu emortuo); note almost masked lipid bodies and persistent encapsulation by a sheath. I: Ascospore in Lugol’s solution, surface view (in statu vivo; lipid bodies not shown); note minute wall ornamentation and persistent encapsulation by a sheath. J: Ascospore from one year old spore print (in statu sicco et vivo) rehydrated by 0.1% KOH; note coalesced polar lipid bodies and persistent encapsulation by a sheath. A-G from CNF-2/9332, H-J from CNF-2/9560. Scale bars = 10 μm. Drawing: N. Matočec. 110 sed and/or forming distinct pustules, but mostly straight and pro- cal, rounded and slightly enlarged at the apex, with spumous com- jecting outwards. Subhymenium thin, of textura intricata. Medullary pound KOH soluble content, without reaction in Lugol’s solution. excipulum of textura intricata, with thin-walled hyphae. Ectal exci- Ascospores ellipsoid, hyaline, appearing eguttulate (in dead state) pulum of vertically oriented textura prismatica, with thin-walled but with small polar aggregations of minute lipid bodies rapidly co- cells, becoming a textura globulosa-subangularis in the outermost alesced into two small polar guttules (in living state), more or less part, then with thick-walled brownish cells. Hairs more or less thick- thick-walled, smooth observed in water and Cotton Blue, but with walled, pale brown, more or less sinuous, septate, obtuse or pointed, a rough to finely verrucose perispore if mounted in Lugol’s solution, with a simple base arising from the globose cells of the outer layer entirely encapsulated by a delicate persistent sheath. Base of apo- of the ectal excipulum. Asci cylindrical, with forked base, arising thecia covered by hyaline anchoring hyphae. from perforated croziers, inamyloid, 8-spored. Paraphyses cylindri- Type species: Lachnea poiraultii Boud.

Plate 4 — Paratricharina poiraultii. Sporogram A: Freshly ejected ascospores in tap water immediatelly after discharge (in statu vivo); note original unaltered bipolar dispersed lipid bodies; equatorially positioned single nucleus also weakly visible. B: Freshly ejected ascospore in tap water about 30 min after discharge (in statu vivo); note bipolar dispersed lipid bodies in a process of coalescence. C: One year old spore print (in statu sicco et vivo) rehydrated by 0.1% KOH; note coalesced polar lipid bodies. D: One year old spore print (in statu sicco et emortuo) rehydrated by 0.1% KOH; note masked polar lipid bodies. E: A group of ascospores from one year old spore print firmly aggregated by capsulate spore sheath (in statu sicco) rehydrated by 0.1% KOH; note that polar lipid bodies are coalesced (in statu vivo) or masked (in statu emortuo). F: Freshly ejected ascospore in CRB im- mediatelly after discharge (in statu vivo); note original unaltered bipolar dispersed lipid bodies; equatorially positioned single nucleus also weakly visible. G: Freshly ejected ascospore in CRB about 30 min after discharge (in statu vivo); note bipolar dispersed lipid bodies in a process of coalescence. H: Freshly ejected ascospores in CR immediatelly after discharge (in statu vivo); note original unaltered bipolar dis- persed lipid bodies and clearly revealed encapsulated sheath causing ascospores to form firm aggregations. I: Ascospores in Lugol’s solution from fresh mount (in statu vivo); note coalesced polar lipid bodies. J: Ascospore in Lugol’s solution from one year old spore print (in statu sicco et vivo); note coalesced polar lipid bodies. K: Ascospore in Lugol’s solution from one year old spore print (in statu sicco et emortuo); note masked polar lipid bodies. L: Ascospore in Cotton Blue/lactic acid from fresh mount (in statu emortuo); note masked polar lipid bodies. M: Ascospores in Cotton Blue/lactic acid from one year old spore print (in statu sicco et emortuo); note masked polar lipid bodies. Red arrows indicate capsulate spore sheath. A-B and F-I from CNF-2/9332, C-E and J-M from CNF-2/9560. Bar = 10 μm. Photos: I. Kušan & N. Matočec. 111 Plate 6 — Paratricharina poiraultii. Microscopic features from type-material A: Ascus and paraphyses. B. Ascospores. C. External hairs. D. Ascospores. E. Marginal hairs. A-C from PC0738324, D-E from PC0738328, all in water mount. Bars = 10 μm. Drawing: N. Van Vooren. 112 Etymology: Paratricharina = composite of Ancient Greek “pará” sel, Göker & Klenk [syn. Geopora pellita (Cooke & Peck) T. Schumach.], which means “beside; next to, near” and “Tricharina”, macro- and mi- Geopora and Tricharina. Morphologically, it shares a similar hymenial croscopically the closest genus to this new one. colour and a similar hairy outer surface with H. pellita, but the latter possesses larger guttulate spores (21–27 μm in length). Humaria sol- In accordance with Art. 55.1 of ICN (Melbourne Code), we propose sequia (Quél.) Van Vooren & Moyne [syn. Humaria aurantiaca (Clem.) the following new combination: Häffner, Benkert & Krisai] also has an orange hymenium and external Paratricharina poiraultii (Boud.) Van Vooren, U. Lindemann, brown hairs, but its spores are narrower and contain larger guttules M. Vega, Ribes, Illescas & Matočec, comb. nov. — MB 812170 (VAN VOOREN & MOYNE, 2010). Among Tricharina species, those with Basionym: Lachnea poiraultii Boud., Bull. Soc. mycol. Fr., 16 (4): 198 yellow-orange coloured hymenium and brown marginal hairs like (1901). T. praecox Chin S. Yang & Korf var. praecox or T. gilva have some macro- and microscopic similarities with Paratricharina poiraultii, Lectotype designated here: PC0738324, “Lachnea Poiraulti” but also clear differences. In contrast toTricharina species, the whole Boud. Icon. mycol. n° 568, Antibes ad terram Februario 1900 misit Poi- outside of the apothecium of P. poiraultii is covered by brown hairs. rault; MBT 201119. Tricharina ascospores contain much smaller amounts of polar gra- nules if any and are devoid of persistent encapsulated sheath. Fur- Phylogenetic analysis thermore, the ectal excipulum of P. poiraultii is composed of textura prismatica becoming a textura globulosa-subangularis of brownish Both inference methods produced very similar topologies, in cells at the 1-3 outmost cell layers, whereas the ectal excipulum of which the same strongly supported clades were recovered. The to- Tricharina species is composed of textura globulosa-angularis (LIN- pology obtained agreed with that reported for the Pyronemataceae DEMANN, 2013). Only Tricharina species with brown marginal hairs by HANSEN et al. (2013). The deeper nodes were not resolved in these have also brownish cells in the ectal excipulum, but these brownish analyses, but the monophyletic status of the following clades was cells are located at the margin whereas the lower flanks of the ectal inferred: Cheilymenia lineage, Pyronema lineage, Octospora lineage, excipulum are composed of hyaline cells. Finally, the mature spores Humaria lineage, Sowerbyella lineage and Scutellinia–Trichophaea of P. poiraultii are thick-walled whereas the spores of Tricharina are lineage (Fig. 1). Within the Scutellinia–Trichophaea lineage, smaller thin-walled at maturity. clades inferred by HANSEN et al. (2013) were also well supported here: Distribution of this species, based on our recent findings and the one formed by Scutellinia, Ramsbottomia and Miladina, another original locality, appears to be in Southern Europe (Fig. 2). Future composed of Trichophaea and Anthracobia, and finally the one com- collections are desirable to confirm this distribution. posed of Tricharina, Geopora, and the recently proposed genus Hoff- mannoscypha (STIELOW et al., 2013). Samples of Lachnea poiraultii Acknowledgements sequenced in the present study were well supported within this last clade, but not supported as a member of any of these three genera. We thank Bart Buyck, curator of the Muséum national d’Histoire Our results infer that the genus Tricharina is paraphyletic (as pre- naturelle (herbarium PC) for the loan of Lachnea poiraultii speci- viously suggested by PERRY et al., 2007). As presently circumscribed, Tricharina is composed of one lineage within the Geopora–Hoffman- mens, Marino Josip Šimić (Umag) for revealing the locality with the noscypha clade and several others of uncertain relationship, inclu- first recent occurrence of Paratricharina poiraultii and for supplying ding T. gilva, the type. Similar paraphyly can be observed in other subsequent fresh collection, Pablo Alvarado (ALVALAB, Santander) groups such as the genus Peziza Fr., where the creation of several for sequencing and analysis, and Javier Bometón (Barcelona) for his new genera to accommodate the different monophyletic lineages data on a recent collection. Thanks are due to João Baptista-Ferreira is advocated (HANSEN et al., 2005b). Hence, in the present study we (Universidade de Lisboa) for willingly opening the way to his pre- choose to propose a new genus for the existing species Lachnea poi- serves in Portugal. Thanks also to Rosanne Healy (Harvard Univer- raultii. sity) for the pre-submission review. Discussion References

Despite remarkable characters, i.e. hairy ascomata, and colour of BARAL H.-O. 1992. — Vital versus herbarium taxonomy: morphologi- hymenium, it is striking that this species has not been found again cal differences between living and dead cells of Ascomycetes, and (or reported) since its discovery in 1900. Indeed, plate 355 from Bou- their taxonomic implications. Mycotaxon, 44: 333-390. dier’s Icones (BOUDIER, 1905-1910) is based on collections previously BOUDIER E. 1885. — Nouvelle classification naturelle des Discomy- cited. Only one reference has been noted in DONADINI (1976: 72) cètes charnus connus généralement sous le nom de Pézizes. Bul- under the name Mycolachnea poiraultii, cited after De Crozals (1861- letin de la Société mycologique de France, 1: 91-120. 1932), a French mycologist who was living in Toulon, a locality close BOUDIER E. 1901 [1900]. — Champignons nouveaux de France. Bulle- to Antibes where Lachnea poiraultii was first collected. Unfortuna- tin de la Société mycologique de France, 16 (4): 193-200 + pl. VIII-IX. tely we were unable to find any data on this putative collection in BOUDIER E. 1905-1910. — Icones mycologicæ, ou Iconographie des De Crozals’ publications. champignons de France. Paris, Paul Klincksieck. 4 vol. In our analyses, Paratricharina poiraultii was strongly supported BOUDIER E. 1907. — Histoire et classification des discomycètes d’Europe. in a clade with Hoffmannoscypha pellita (Cooke & Peck) Stielow, Hen- Paris, Paul Klincksieck, 222 p.

Table 1 — Collections of Paratricharina poiraultii sequenced and used in the molecular phylogenetic study, with voucher information and GenBank accession numbers. The abbreviation in the “Herb.-No.” indicates the private herbaria of Uwe Lindemann (= U.L.)

Species Herb.-No. Country, Collecting Date, Collector 28S ITS tef-1α rpb2 P. poiraultii U.L.178-14 Portugal, 2014, M. Vega KP052789 KP052788 KP052790 KP052791 P. poiraultii U.L.179-14 Spain, 2014, T. Illescas, M. Á. Ribes et al. KP052785 KP052784 KP052786 KP052787 113 Fig. 1 — Consensus 28S-tef 1α-rpb2 phylogram of the family Pyronemataceae obtained in MrBayes 3.1 from 1,950 sampled trees. Nodes significantly supported by both Bayesian and ML analyses are highlighted with bold bars (0.95 PP, 65 BP). Nodes supported by just one of these inference methods are annotated with labels representing their actual posterior probabilities (left) and ML bootstrap proportions (right). (*) This sequence appears in GenBank as Geopora pellita. 114 Fig. 2 — Distribution map of known collections of Paratricharina poiraultii

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Nicolas Van Vooren Uwe Lindemann Marcel Vega 36 rue de la Garde Pflügerstrasse 62 Kohlhoefen 17 69005 Lyon 12047 Berlin 20355 Hamburg France Germany Germany [email protected] [email protected]

Miguel Ángel Ribes Tomás Illescas Neven Matočec Avda. Pablo Neruda 120 El Avellano 3 Ruđer Bošković Institute, Bijenička 54 28018 Madrid 14006 Córdoba 10000 Zagreb Spain Spain Croatia [email protected] [email protected] [email protected]

Ivana Kušan Ruđer Bošković Institute, Bijenička 54 10000 Zagreb Croatia [email protected]

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